Chromosome aberrations in bone marrow cells of rats treated with

MTBE

Iman Abd El-Moneim Darwish and Sahar Abd El-Razik Mosallam

Zoology Department ,Women, s College for Arts, Science and Education, Ain Shams University, Cairo, Egypt

Abstract: In the present study, genotoxic effect of methyl tert butyl ether MTBE was analyzed by measuring
chromosomal aberrations (CAs) in bone marrow cells of rats. Rats administered MTBE orally at 800, 1600mg/kg/day in
corn oil for 14 and 28 consecutive days. Control rats received injection of distilled water. An additional two groups of
rats received corn oil and served as vehicle controls. Treatment of corn oil for 14 and 28 days failed to induce
chromosomal aberrations. The highest percentage of chromosomal aberrations was produced by the two tested dose 14
days after treatment. The most structural aberrations were Robertsonion translocations, deletion, dicentric, end to end
association while, ring, acentric fragment and gaps were rare. The present results indicate that MTBE is harmful to
mammalian genetic material.

Keywords: Methyl tert butyl ether (MTBE), chromosomal aberrations assay (CAA), male rats Rattus norvegicus.

INTRODUCTION methyl tertiary-butyl ether (MTBE) in Salmonella

Methyl tert-butyl ether (MTBE) is a well-known
environmental contaminant owing to its high solubility in
water (Sawunyama and Bailey, 2002). MTBE is widely
used as an additive to gasoline, to increase oxygen content
and reduce tailpipe emission of carbon monoxide
(Perbellini et al., 2003). Since MTBE is resistant to most
physical methods of treating fuel-contaminated water,
biodegradation may be a useful means of remediation
(Youngster et al., 2008). MTBE had potential uses as an
anti-angiogenic treatment for solid tumors with minimal
toxicity and constitutes a good solvent for mixed
cholesterol stones (Kozlosky et al., 2013 and Dai et al.,
1988). MTBE-induced vascular toxicity in zebrafish
(Danio rerio), embryos (Bonventre et al., 2011); induced
cell injury, associated with mitochondrial dysfunction,
and alterations in cytosolic Ca2+ in isolated rabbit tracheal
epithelial cells (Wang et al., 2008); resulted in mild
changes in hormone levels and endocrine-sensitive tissues
in rats (Williams et al., 2000) and caused an increased
incidence of Leydig cell testicular tumors in male rats, a
dose-related increase of leukemias, an increase of
dysplastic proliferations of lymphoreticular tissues and
also an increase of uterine sarcomas in females (Belpoggi
et al., 1997).
However, Werner et al. (2001) suggested that at
environmental MTBE exposure levels found in surface
waters (<0.1 mg/l), this compound was likely not acutely
toxic to aquatic life.
Moreover, MTBE could induce DNA double-strand
breaks and inhibit cell growth in vitro (Song et al., 2002
and Chen et al., 2008). Also, the mutagenic effects of
*Corresponding author: e-mail: imandarwish73@yahoo.com
Pak. J. Pharm. Sci., Vol.----, No.0, ---------- 20----, pp.000-000
typhimurium TA102, in the presence of an exogenous
metabolic activation system could not be confirmed
(McGregor et al., 2005).
The present study aimed to investigate the chromosomal
aberrations induced in rat’s bone marrow cells under the
effect of MTBE.
MATERIALS AND METHODS
Chemical
Methyl tert-Butyl ether (MTBE) was obtained from
Sigma Chemical Company. MTBE Cas Number: 1634-
04-4; Formula: C5H12O; Molecular weight: 88.15; melting
point: -109oC; boiling point: 55.2oC; density: 0.74,
Refractive Index: 1.3689 and the purity was more than
99% (GC≥99.9%). The compound is highly volatile (US
EPA, 1997). The structural formula is showed in fig. (1)
as stated by Belpoggi et al. (1995).
Animals
Adult male rats aged (50-100) weeks and weighing 50-
70g were employed. The animals were maintained under
proper environmental conditions, i.e. temperature 25±2oC,
humidity 50±5% with a 12h light/dark period. They were
housed in cages and fed with pelleted standard diet and
tap water ad libitum. The animals were allowed to
acclimatize for at least 5 days prior to the study. Six mice
were used for each dose and each sampling time.
1
Chromosome aberrations in bone marrow cells of rats treated with MTBE

Treatment 1600 mg MTBE decreased from 16±6.06% after 14 days

The rats were divided into seven groups treated via oral
gavage. The first group (control) treated with distilled
water. Groups 2, 3 & 4 received corn oil, 800mg/kg b.w.
and1600mg/kg b. w. for 14 days, respectively. Groups 5, 6
& 7 received corn oil, 800mg/kg b.w. and 1600mg/kg b.
w. for 28 days, respectively. These doses were chosen
according to Li et al. (2008). The vehicle was corn oil.
Dilution of MTBE in corn oil was made before the start of
the test and each day prior to test renewal in a 5ml
volumetric flask. Rats were tested to ensure that there was
no evidence of infectious pathogens.
to 13.33±7.11% after 28 days. The chromosomal
aberrations percentages in rats was reached 13±5.17 after
oral administration of MTBE at 800 mg for 14
consecutive days. This percentage decreased to 10±6.19
in bone-marrow cells after 28 days and only 1.66% of
them had chromatid deletions (table 1). The most
structural aberrations were Robertsonian translocation,
dicentric and deletions, while ring, gap and acentric
fragments were similar in number. Other aberrations such
as breaks were not spotted. Deleted chromosomes
occurred in 2.33% of the examined metaphases 14 days
post-treatment with the small dose. Chromosomal
exchanges were also observed at certain instances.
Acentric fragments occurred as small parts of
chromosomes, each having no apparent centromere.
Fragments were single or double, fig. (2). It is of interest
to state that, some metaphase spreads might have more
than one type of structural aberrations. Cells with more
than one aberrations were grouped as multiple
aberrations.

Fig. 1: Structural chromosomal aberrations in bone

marrow cells induced by MTBE via the oral gavage route

Chromosome aberration studies

For chromosomal aberrations, rats at all fixation intervals
were injected intraperitoneally with 0.04% colchicine
solution, 0.5 ml/10g body weight 2h prior to sacrifice.
Bone marrow from the femur was flushed out into 0.56%
KCl hypotonic solution. Cells were centrifuged and fixed
with two or three changes of Carnoy’s fixative (3: 1
methanol- acetic acid). The slides were prepared by
flame-drying and stained with buffered Giemsa (pH 6.8).
Fifty metaphase spreads per animal were analyzed for
chromosomal aberrations (Tice et al., 1994).

STATISTICAL ANALYSIS

Statistical significant was evaluated using an ANOVA

(one-way) test following by Tukey test. Statistical Fig. 2: Structural chromosomal aberrations in bone
significance was accepted if p≤ 0.05. marrow cells induced by MTBE via the oral gavage rout.
R t= Robertsonian translocation, e to e a= end to end
RESULTS association, d= deletion,
dic= dicentric, ac f= acentric fragment, r= ring, g= gap
Chromosome aberrations assay: Among control group, the
DISCUSSION
total chromosomal aberrations were spotted on 5±3.28%
metaphases from 300 metaphases. The frequencies of
Rats were assessed at two intervals, against suitable
chromosome aberrations were not significantly increased
controls, using parameters like chromosomal aberrations.
in rats treated with corn oil for 14 or 28 days (table 1).
Because a previous study demonstrated a decrease in
The highest percentage of aberrations was produced by
plasma testosterone in male Sprague-Dawley rats treated
the two tested dose 14 days after treatment. A higher level
orally with 800 mg MTBE /kg/day for 28 days (Day et
of chromosomal aberrations was found at the highest
al., 1998), a 28-day MTBE treatment was used along with
concentration of MTBE for 14 days. The percentage
a 15-day treatment to compare possible length of
structural chromosomal aberrations for rats treated with

2 Pak. J. Pharm. Sci., Vol.----, No.0, ---------- 20----, pp.000-000

 Iman Abd El-Moneim Darwish and Sahar Abd El-Razik Mosallam

exposure differences. They indicated that MTBE alters concentration exposures (0.52 and 0.63 hours,
endocrine-sensitive parameters in adult male rats. respectively). After the repeat exposures, the MTBE t1/2
Table 1: The percentage of the different types of structural chromosomal aberrations in male rats treated with methyl tert
butyl ether

Number of Structural chromosomal aberrations/300 cells

Experimental
Duration examined Total Mean ±S.D%
group R.t. e to e ass del dic ac.f. r. g
metaphases
control 0 300 2 1 1 6 2 1 2 15 5.±3.28
Corn oil 300 3 1 2 5 4 4 1 20 6.66±1.63-
800mg/kg b.w. 14 days 300 12 6 7 7 3 4 - 39 13±5.17-
1600mg/kg b.w. 300 12 2 9 11 4 6 4 48 16±6.06*,**
Corn oil 300 3 4 5 3 1 4 2 22 7.33±3.26-
800mg/kg b.w. 28 days 300 5 10 5 4 1 - 5 30 10±6.19-
1600mg/kg b.w. 300 7 5 9 6 5 4 4 40 13.33±7.11-
SCA=Structural chromosomal aberrations, R t = Robertsonian translocations, e to e a= end to end association, d= deletion, dic=
dicentric, ac f =acentric fragment, r= ring, g = gap, SCA=Structural chromosomal aberrations, - = non significant p≥0.05 , *p≤0.05
versus corn oil group, **p≤0.01 versus control group
MTBE clearly induced chromosome aberrations under the was slightly shorter 0.48 and 0.51 hours, respectively
treatment conditions. It has also been shown in the present (Integrated Risk Information System, IRIS 1991).
study that chromosomal aberrations (CAs) induction Between 35 and 69% of the MTBE retained after the end
increases in dose-dependent manner. The present of the exposure was recovered as metabolites in urine of
observations were agreements with the results obtained by both humans and rats (Amberg et al., 1999).
Li et al. (2008). They suggested that relatively high dose
of MTBE could be exerted reproductive system toxicity Moreover, it is possible that MTBE could also express an
of male rats and disturbed the secretion of testosterone, aneugenic mode of action as inhibiting cell division and
luteinizing hormone and follicle stimulating hormone, mitotic spindle apparatus. The chromosomal aberrations
possibly due to oxidative stress induced by treatment of observed in animals clearly indicate that this compound
MTBE for 2 weeks. interacts with chromatin DNA and induce damage there
(data not published).
Corn oil alone did not induce chromosomal damage in
bone marrow cells. The induction of chromosome Statistically significant chromosomal aberrations,
aberrations was significantly increased in rats after micronucleus and sperm abnormalities revealed the
multiple oral treatments of 1600 mg MTBE Kg-1b.w. The genotoxicity of a test compound as stated by Jayashree et
chromosomal aberrations percentage occurred at a higher al. (1994). Similarly, chromosomal damage is considered
rate after 14 days of 1600mg MTBE /kg b.w. The results to detect early effects of xenobiotic insults and elevation
demonstrated that the number of abnormal metaphases of the frequency of CAs is a sensitive cytogenetic assay
identified in the 14 day treatments is higher than that for detecting exposure to mutagens and carcinogens
observed in the 28 day treatment, for the two treated (Bonassi et al., 1995; Sierra-Tores et al., 1998).
group. As suggested by Gautam and Kapoor (1991) such
reduction may be due to the fact that cells with severe Robertsonian translocations, deletions and dicentric were
chromosomal damage might have been deleted in cell increased at two doses studied indicating the clastogenic
cycle following the treatment. Then the level of potential of MTBE. These findings suggest that G1 could
chromosomal aberrations decreased after 28 days of be the cell cycle phase most sensitive to MTBE genetic
MTBE treatment, although still higher than the control damage. MTBE could induce the higher expression of c-
level. The observed cytological aberrations in the bone mycprotooncogene, which suggested it could promote cell
marrow assay reveal that MTBE has the potentiality to proliferation-one of possible mechanisms of
induce genotoxicity at the chromosome level. carcinogenesis in animals (Zhou et al., 1999).

To the author′s opinion, the drug causes chromosome
aberration which seems to be reversible as the drug
eliminated or diminished from the bone marrow through
the metabolic process. Such level of chromosome
aberrations might be related to the usual decrease of DNA
adduct in rats. The elimination half-life (t1/2) of MTBE
was approximately the same after single low-and high-
Giannotti et al. (2002) mentioned that strand breaks are
too short lived to allow detection after a 3h treatment
period (due to preferential repair), indicating the need for
shorter exposure times in some cases to optimize their
detection.

Pak. J. Pharm. Sci., Vol.----, No.0, ---------- 20----, pp.000-000 3

Chromosome aberrations in bone marrow cells of rats treated with MTBE
Chromosomal aberrations qualitatively and quantitatively
detect clastogenic activity, while the micronucleus assay
detects both clastogenic effects and damage to the mitotic
apparatus, some of which might have aneugenic
consequences (Dimitrov et al., 2006).
Moreover, several authors reported the genotoxic effects
of MTBE in vivo and/or in vitro. Accordingly, Williams-
Hill et al. (1999) concluded that MTBE and its
metabolites induce a mutagenic pathway involving
oxidation of DNA bases and an intact repair system.
Yuan et al. (2007) concluded that the methyl group of
MTBE and tert-butyl alcohol definitely form adducts with
DNA in mouse liver, lung and kidney. The methyl group
of MTBE is the predominant binding part in liver, while
the methyl group and the tert-butyl groups give
comparable contributions to the adduct formation in lung
and kidney. Bonventre et al. (2011) observed significant
decrease in the expression of vegfa, vegfc, and flk1/kdr in
vascular development following embryonic exposure to
MTBE.
Also, Ghasemi and Ahmadi (2014) revealed that MTBE
may have interaction with calf thymus DNA (ct-DNA) via
the minor groove of DNA. Also, MTBE may be
complexed into the basket of G-quadruplex structure.
Recently, Bravo et al. (2015) reported that the expression
of alkane monooxygenase (alkB) gene was related to the
co-metabolic oxidation of MTBE. Valipour et al. (2015)
showed that insulin formed a molten globule (MG)-like
structure in the presence of 8uM MTBE due to protein
oxidation and reactive oxygen specious (ROS) generation.
CONCLUSION
The present results demonstrated that MTBE has a
clastogenic potential as measured by the bone marrow
chromosomal aberrations in rats.
Acknowledgement : We would like to express our
gratitude to Prof. dr. Karima Mohammad Sweify for
critical comments on this research and manuscript.
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